Cloning,expression and antibacterial function analysis of novel antibacterial peptide gene ALF-like in Penaeus monodon

【Objective】To clone a novel antibacterial peptide gene from Penaeus monodon(PmALF-like),and to clarify its structural characteristics,tissue expression pattern,and its expression changes after stimulation of Vibrio parahemolyticus,so as to explore its role and function in antibacterial innate immunity,with an aim to provide reference for elucidating the disease-resistant immune mechanism of P. monodon. 【Method】Based on the expression sequence target(EST) sequence of PmALF-like gene, the full-length cDNA sequence of PmALF-like was obtained by RACE amplification. Bioinformatics analysis was performed using online softwares such as ProtParam,SignalP 5.0,MegAlign and MEGA 7.0. The expression characteristics of PmALF-like gene in different tissues and its expression changes after V. parahemolyticus stimulation were detected by real-time fluorescence quantitative PCR(qRT-PCR). The recombinant protein PmALF-like or mature peptide was obtained by prokaryotic recombination method(removing signaling peptide),and the inhibitory effect of PmALF-like mature peptide on bacterial growth was investigated by liquid medium method and Oxford cup method. 【Result】The total length of the cDNA sequence of PmALF-like gene was 1242 bp,including the 5' non-coding region(5'-UTR) of 45 bp,the 3' non-coding region(3'-UTR) of 798 bp and the open reading frame(ORF) of 399 bp,encoding 132 amino acids. The molecular weight was 15.26 kD and the theoretical isoelectric point(p I) was 7.67. A signal peptide of PmALF-like protein was predicted by the online software SignalP 5.0 with a splicing site at the 18^th amino acid.The results of qRT-PCR showed that PmALF-like was expressed in hemopancreas,hepatopancreas,gills,intestinal tract,muscle and other tissues to varying degrees,with the highest relative expression level in hepatopancreas,followed by hemopancreas,and less relative expression level in muscle tissue. After stimulation by V. parahemolyticus,the expression level of PmALF-like gene in hepatopancreas of P. monodon produced immune response,and its gene expression was significantly(P<0.05) or extremely significantly(P<0.01) up-regulated at 6-72 h. PmALF-like mature peptide without signal peptide was successfully obtained by prokaryotic expression method in vitro. It was found that the mature peptide of PmALF-like had obvious inhibitory effect on the growth of Aeromonas victori,V. parahemolyticus and Aeromonas hydrophila by liquid medium method and Oxford cup method. 【Conclusion】PmALF-like gene is widely expressed in various tissues of P. maculatus, its mature peptide has obvious inhibitory effects on the growth of A. vickeri,V. parahaemolyticus and A. hydrophila.